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. 2017 Aug 21;7:8328. doi: 10.1038/s41598-017-08270-w

Figure 3.

Figure 3

Accuracy of the Selfie-dPCR method for quantifying absolute quantities of synthetic RNA and DNA by using two different amplicons in the same target template. (A) Schematic diagram showing the locations of the two amplicons within the synthetic template of mouse Gsk3β RNA and DNA and illustrating Selfie-dPCR amplification of RNA + DNA in the RT+ reaction and only DNA in the RT− reaction for each amplicon. (B) Scatter plot showing droplet digital PCR analysis of Gsk3β DNA + RNA (RT+) and Gsk3β DNA (RT−) for Gsk3β-86 (left two panels) and Gsk3β-81 (right two panels) in a sample containing both synthetic DNA and RNA. Blue dots indicate droplets containing target template for which end-point PCR has produced an amplicon. Selfie-dPCR calculation shows that the numbers of Gsk3β transcripts per gene obtained for each amplicon are not significantly different (p > 0.05, n = 3). (C–E) Accuracy of Selfie-dPCR assessed by measuring known amounts of RNA (C), DNA (D) and combined DNA + RNA (E) in RT+ and RT− reactions using two different amplicons (Gsk3β-86 and Gsk3β-81). No significant differences between the amplicons were found in the RNA, DNA and DNA-RNA copies detected, thus indicating that absolute measurement of nucleic acid copy numbers with Selfie-PCR is independent of the targeted region in the template sequence (p > 0.05, n = 3).